GABA Pathway Rate-Limit Citrate Degradation in Postharvest Citrus

Article pubs.acs.org/JAFC

GABA Pathway Rate-Limit Citrate Degradation in Postharvest Citrus Fruit Evidence from HB Pumelo (Citrus grandis) × Fairchild (Citrus reticulata) Hybrid Population Ling Sheng, Dandan Shen, Wei Yang, Mingfei Zhang, Yunliu Zeng, Juan Xu, Xiuxin Deng, and Yunjiang Cheng* Key Laboratory of Horticultural Plant Biology (Ministry of Education), Key Laboratory of Horticultural Crop Biology and Genetic Improvement (Central Region), MOA, PR China, College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China S Supporting Information *

ABSTRACT: Organic acids are a major index of fresh fruit marketing properties. However, the genetic effects on the organic acid level in postharvest citrus fruit still remain unknown. Here, we used the fruits of about 40 lines in a hybrid population (highacid “HB Pumelo” × low-acid “Fairchild”) to analyze the organic acid metabolism of postharvest citrus fruit. A transgressive content of titratable acid (TA) was observed, which was attributed to citrate accumulation. High- and low-acid fruits (No. 130, 168 and No. 080, 181, respectively) were chosen for further study. Gene expression analysis on citrate metabolism showed that the high accumulation of citrate could be attributed to the low activity of γ-aminobutyric acid (GABA) shunt, and was partially due to the block of tricarboxylic acid (TCA) cycle by low mitochondrial aconitase (m-ACO) expression. TA level was significantly negatively correlated with weight loss in fruits during postharvest storage, implying a close relationship between organic acid and water metabolism. KEYWORDS: citrate, gene expression, fruit water loss, postharvest storage, citrus hybrid population

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INTRODUCTION Quality of horticultural products comprises several aspects, including appearance, texture, flavor, and nutritional value, particularly the sensory issues, which have become a primary factor for consumer’s acceptance of the fruits.1 Organic acids (mainly including citrate and malate), soluble sugars (mainly including fructose, glucose and sucrose), and amino acids are the most important compounds for the flavor and nutrition of citrus fruit. Previous studies have shown that organic acids also play an important role in extending the shelf life of fresh fruits and the processed products,2 which may be related to their participation in fruit senescence process.3,4 Hence, the consumption of organic acids during postharvest storage is a key factor affecting the fruit flavor and storage performance. In recent years, organic acid metabolism in fruit cells has attracted more and more research attention. As the main organic acid in citrus fruit, citrate accounts for 70−90% of the total organic acids,5 and its content is determined by the balance between its synthesis, catabolism, transport, and vacuolar storage.6−8 Citrate synthase (CS) may not be responsible for the differences in acidity among citrus varieties.9−11 The inhibition of mitochondrial aconitase (mACO) is the prerequisite for the transport of citrate into cytoplasm, which could result in citrate accumulation.11,12 Previous studies have indicated that citrate is accumulated during fruit development and then reduced during fruit ripening and postharvest storage.6,8 γ-Aminobutyric acid (GABA) shunt is one of the major pathways that participates in citrate utilization during fruit ripening.8 The short pathway starts with the irreversible decarboxylation of glutamate and © 2017 American Chemical Society

consumption of protons to produce GABA and CO2 by glutamate decarboxylase (GAD) in the cytosol. GABA is then transported to the mitochondria by GABA permease (GABP), where it is converted to succinate by GABA transaminase (GABA-T) and succinate semialdehyde dehydrogenase (SSADH).13,14 GAD is the key enzyme in this process and is closely related to citrate utilization.15,16 A recent study showed that dicarboxylate carrier gene (DIC) is involved in citrate degradation during fruit development and postharvest hot-air triggered citrate reduction.17 Besides, some researchers demonstrated that a bHLH transcription factor gene (TT8)18 and the amino acid transporter family protein (AA-TFP)6 are also correlated with citrate metabolism. The contents of organic acids vary greatly among different citrus varieties. In general, HB Pumelo (HB, Citrus grandis) belongs to the high-acid type; and some Mandarin varieties belong to the low-acid type, such as Fairchild (FC, Citrus reticulata). Fruit inner quality, including the content of organic acids, is affected by genetics, environmental factors, and cultural practices.19−22 Most previous studies were focused on the effects of cultivation techniques and environment factors on fruit quality.20,22,23 However, there have been very few studies about the effect of genetic background on postharvest fruit quality traits. In this work, we generated a hybrid population by the cross of HB with FC conducted 10 years ago. High- and Received: Revised: Accepted: Published: 1669

November 21, 2016 December 27, 2016 February 2, 2017 February 2, 2017 DOI: 10.1021/acs.jafc.6b05237 J. Agric. Food Chem. 2017, 65, 1669−1676

Article

Journal of Agricultural and Food Chemistry

Figure 1. Content of TA in HB × Fairchild hybrid population in 2014 (A). The gray column represents the selected fruits for storage experiment. The selected fruits are shown in (B). Vertical bars represent the standard errors of the means.

low-acid fruits (No. 130, 168 and No. 080, 181, respectively) were selected to compare the difference in organic acid accumulation during postharvest storage, aiming to analyze the effect of genetic background on organic acid metabolism in citrus fruit.

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CI =

1000a Lb

Titratable acid (TA, %) was measured by a digital acidity meter (GMK-835, G-WON HITECH CO., LTD, Korea) following the manufacturer’s instructions.24 Total soluble solid (TSS, %) and weight loss (%) were measured according to the method described previously.25 TSS was determined with a refractometer (Model: Pocket PAL-1, Atago Inc., Toyko, Japan) according to the manufacturer’s instructions. The 2, 6-dichloroindophenol titrimetric method was used to determine the vitamin C (VC) content of pulp extracts as previously described.26 Extraction and titration were performed in triplicates. The results were expressed in mg ascorbic acid per 100 g fresh weight (mg/ 100g FW). Respiration Rate Determination. The respiration rate (mgCO2/ kg/h) of fruit was measured by analyzing the headspace gas composition according to the method described before.27 Two fruits (with three replicates) were weighed and sealed in a 2.6-L preserving box at 25 °C for 2 h. Headspace gas samples were withdrawn with a 1 mL syringe. The CO2 concentration in the gas samples was determined with a gas chromatography (Gas Chromatography, GC; Agilent, 7890A, USA) equipped with a thermal conductivity detector and a CTR-1 column (Alltech Associates, USA). Primary Metabolite Analysis by GC-MS. Primary metabolites were detected with three biological replicates through GC-MS analysis as described previously.3 Exactly 0.2 g of mixed sample was ground in liquid nitrogen, and then extracted with 2.7 mL of chromatographic grade methanol, followed by the addition of 300 μL of internal standard (0.2 mg/mL ribitol). After drastic shaking and ultrasonic treatment for 30 min, the mixture was put into a thermostatic water bath at 70 °C for 15 min and centrifuged at 5000 g for 15 min. Then, 100 μL supernatant was obtained and vacuum-concentrated.

MATERIALS AND METHODS

Plant Materials. Commercially mature fruits of HB Pumelo (Citrus grandis) and Fairchild (Citrus reticulata) and the fruits of their hybrid population were harvested from the orchard in Huazhong Agricultural University (Hubei Province, China) in 2012, 2013, and 2014. After harvest, the fruits without any physical damages and uniform in size were randomly selected for further experiments. For postharvest storage experiments in 2014, the fruits with the lowest acid (No. 130 and 168) and the highest acid (No. 080 and 181) were chosen, and were unipacked and stored at room temperature (20 ± 3 °C) with a relative humidity of 85−90% for about three months. To measure the levels of organic acids, sugars, and amino acids and the expression of related genes, juice sacs were separated from 5 fruits in each individual plant at 0, 20, 40, 60, and 85 days after storage (DAS) with three replicates. Samples were immediately frozen in liquid nitrogen and stored at −80 °C until analysis. Quality Parameter Determination. The fruit weight was measured using an electronic hydrostatic balance (Model: MP31001, Shanghai Selon Scientific Instrument, Co., Ltd., Shanghai, China) with an accuracy of ±0.01 g. Fruit shape index was calculated by longitudinal diameter divided by transverse diameter. Fruit surface color was measured as previously described.3 Three readings were taken at different points around the equatorial region of each fruit using a Konica Minolta Sensing CM-5 Spectrophotometer (Japan), which provides CIE L*, a*, and b* values. The color index (CI) was calculated by the equation: 1670

DOI: 10.1021/acs.jafc.6b05237 J. Agric. Food Chem. 2017, 65, 1669−1676

Article


Figure 2. Changes of TA (A) and TSS (B) content in high- and low-acid fruits during postharvest storage. Vertical bars represent the standard errors of the means. Values with different letters within the same figure are significantly different according to t-test at p < 0.05.

Figure 3. Changes of citrate (A) and malate (B) content in high- and low-acid fruits during postharvest storage. Vertical bars represent the standard errors of the means. Values with different letters within the same figure are significantly different according to t-test at p < 0.05. Prior to GC−MS analysis, metabolites were derivatized through dissolving in 80 μL methoxamine hydrochloride (20 mg/mL in pyridine), incubated for 90 min at 37 °C, and then reacted with 80 μL of MSTFA (N-methyl-N-(trimethylsilyl) trifluoroacetamide) for 30 min at 37 °C. After microporous filtering, the samples were analyzed by GC−MS. Exactly 1 μL of each sample was injected into the gas chromatograph onto a fused-silica capillary column (30 m × 0.25 mm i.d., 0.25 μm DB-5 MS stationary phase). The injector temperature was 250 °C, and the carrier gas was at a flow rate of 1.0 mL/min. The column temperature was held at 100 °C for 1 min, increased to 184 °C with a temperature gradient of 3 °C/min, increased to 190 °C at 0.5 °C/min for 1 min and increased to 280 °C at 15 °C/min for 5 min. The flow rate of carrier helium (99.999%) gas was 1 mL/min. The MS operating parameters were as follows: 70 eV ionization voltage, 200 °C ion source temperature, and 250 °C interface temperature. Total ion current spectra were recorded over a mass range of m/z 45−600 in scan mode. For individual metabolites, we quantified the final concentrations (μg/g FW) using a ribitol internal standard. The final data were used for statistical analyses. RNA Isolation and Real-Time Quantitative PCR. Total RNA of each sample was independently isolated according to the method described previously,28 and was used for real-time quantitative PCR. The related genes were selected and validated by real-time quantitative PCR (qRT-PCR). The RNA samples were reverse transcribed using a ReverAidTM M-MuIV reverse transcriptase Kit (MBI, Lithuania). The gene encoding actin was used as the endogenous control according to the previous study.29 The specific primer pairs of the selected genes were designed using Primer Express 3.0 (Applied Biosystems, Foster City, CA), and are listed in Table S2. Statistical Analysis. All data were analyzed using one-way analysis of variance (ANOVA) and expressed as average ± SE. Significant differences were considered at p < 0.05. Pearson’s correlation analysis was performed using the SAS V8.0 software package (SAS Institute, Cary, NC, USA). The correlation analysis was performed among various parameters with standardization procedure.

weight, fruit shape, pericarp thickness, fruit surface color, TSS, VC, and TA of the hybrid population, were traced for three years. A stable transgressive level of TA content was observed in the mature fruits (Figure 1A and Figure S1). Such transgressive behavior was not observed for other fruit quality traits, such as fruit weight, pericarp thickness, surface color, TSS, and VC content (Table S1). We then focused our research on TA, its content of the fruit ranged from 1.96 ± 0.04% to 6.87 ± 0.11% (Figure 1A). Two prolific hybrid plants exhibiting a stable phenotype of low acid (No. 130 and No. 168) and high acid (No. 080 and No. 181) were respectively chosen for further analysis (Figure 1B). Changes of TA and TSS Content in High- and LowAcid Fruits during Postharvest Storage. The TA content generally showed a decreasing trend during postharvest storage, especially from 20 to 40 DAS in high-acid fruits (Figure 2A). It was obvious that the trait of significantly higher acid was maintained throughout the storage period. Unlike that of TA, the content of TSS was maintained at a relatively stable level during storage (Figure 2B). On the whole, the TSS content in high-acid fruits was lower than that in low-acid ones, but the difference was not as remarkable as that in TA content. It could be inferred that the flavor quality decline of citrus fruit during postharvest storage is primarily due to the decrease of TA. Hence, we further analyzed the changes in the contents of the major organic acids in high- and low-acid citrus fruits during postharvest storage. Changes of Organic Acid Content in High- and LowAcid Fruits during Postharvest Storage. Citrate and malate are the major organic acids in citrus fruit. Our results indicated that the transgressive level was mainly dependent on citrate, not on malate (data not shown). The citrate content in mature high-acid fruits (20401.58 ± 550.98 and 13899.40 ± 423.26 μg/g FW, respectively, in No. 080 and 181) was significantly higher than that in low-acid fruits (6574.35 ± 46.50 and 9592.5 ± 175.80 μg/g FW, respectively, in No. 130 and 168) (0 DAS, Figure 3A), which was consistently observed throughout the whole storage period (Figure 3A). It is worth noting that a

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RESULTS Characteristics of Mature Fruit of HB × Fairchild Hybrid Population. The quality characteristics, including fruit 1671


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Figure 4. Changes of sugar (A−C) and amino acid (D−L) content in high- and low-acid fruits during postharvest storage. Vertical bars represent the standard errors of the means. Values with different letters within the same figure are significantly different according to t-test at p < 0.05.

soluble sugars had positive correlation with sucrose in No. 130 and 181 (data not shown). The contents of amino acids did not follow a certain pattern as the content of citrate, except for that of aspartate and asparagine (Figure 4D−L). Aspartate was higher in high-acid fruits than in low-acid fruits during storage (Figure 4F), while it was the opposite case for asparagine (Figure 4G). The contents of alanine, valine, serine, threonine, and proline were all relatively higher in the fruits of No. 168 and 080 (Figure 4 H− L). There was an increasing tendency of GABA content along with the extension of storage time (Figure 4D). It was always the lowest in the fruits of No. 130 during storage, and was strikingly increased at 85 DAS in the fruits of No. 168 and 181. The content of glutamate showed irregular variation in highand low-acid fruits during storage, but it was obviously lower in high-acid fruits than in low-acid ones at 40 DAS (Figure 4E). In summary, the characteristics of the contents of these metabolites at mature stage (0 DAS) were consistently maintained throughout the storage period (Figure 4). Expression of Citrate Metabolism-Related Genes in High- and Low-Acid Fruits during Postharvest Storage. In order to understand the accumulation mechanism of citrate in high-acid fruits, we detected the expression of the genes related to citrate metabolism. As can be seen in Figure 5, the expression of CS was not high in high-acid fruits during storage as expected (Figure 5A). Importantly, the expression of mACO was significantly lower in high-acid fruits than in low-acid

significant increase of citrate content occurred in No. 130 and No. 181 at 85 DAS compared with at 0 DAS; however, in No. 168 and No. 080 fruits, the citrate content showed fluctuating decline during storage (Figure 3A). In general, the content of malate showed an obviously decreasing tendency during storage, especially in No. 130, 080, and 181 fruit (Figure 3B), which could explain the decrease of TA content in No. 130 and 181 while the citrate content in the two lines was maintained during storage; besides, unlike that of citrate, the content of malate did not follow a particular pattern. Therefore, we focused on the accumulation mechanism of citrate in high-acid fruits in the following analyses. Changes of Sugar and Amino Acid Contents in Highand Low-Acid Fruits during Postharvest Storage. Organic acid metabolism is closely connected with sugar and amino acid metabolism. Thus, we analyzed the changes of sugars and amino acids in high- and low-acid fruits during postharvest storage. Fructose, glucose, and sucrose are the main soluble sugars in citrus fruits. As can be seen in Figure 4, the contents of fructose, glucose, and sucrose were lower in highacid fruits than in low-acid fruits during storage, especially in No. 181 (Figure 4A, B, and C). In addition, the contents of fructose and glucose showed a gradually increasing trend from 40 to 85 DAS. Correlation analysis indicated that the content variation of fructose was significantly positively correlated with that of glucose in both high- and low-acid fruits, and these two 1672


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Figure 5. Expression of the genes related to citrate metabolism. (A) CS (citrate synthase); (B) m-ACO (mitochondrion aconitase); (C) NAD-IDH (NAD-isocitrate dehydrogenase); (D) 2-OGDH (2-oxoglutarate dehydrogenase); (E) GAD1 (Glutamate decarboxylase 1); (F) GABP (mitochondrial GABA permease); (G) GABA-T (GABA transaminase); (H) SSADH (succinic semialdehyde dehydrogenase); (I) DIC (dicarboxylate carrier gene); (J) AA-TFP (amino acids transporter family protein); (K) TT8 (bHLH transcription factor family gene); (L) AHA10 (H+-ATPase Isoform 10). Vertical bars represent the standard errors of the means. Values with different letters within the same figure are significantly different according to t-test at p < 0.05.

Figure 6. Respiration rate (A) and fruit weight loss (B) in high- and low-acid fruits during postharvest storage. Vertical bars represent the standard errors of the means. Values with different letters within the same figure are significantly different according to t-test at p < 0.05.

especially from 40 to 85 DAS (Figure 5J and L). TT8, a bHLH family gene, had high expression in high-acid fruits mainly at 40 and 60 DAS (Figure 5K). It is worth noting that there was a significant increase of DIC expression from 60 to 85 DAS in No. 181, and citrate was also strikingly increased in this period (Figure 3A and Figure 5I). These results suggested that the high citrate accumulation in high-acid fruits could be attributed to the low activity of citrate degradation pathway during fruit postharvest storage. Respiration Rate and Fruit Water Loss in High- and Low-Acid Fruits during Postharvest Storage. The respiration rate showed a gradually decreasing tendency during storage in all samples, and was significantly lower in high-acid fruits than in low-acid fruits (Figure 6A). During postharvest storage, the fruit weight loss (mainly water loss) showed a gradually increasing tendency in both high- and low-acid fruits (Figure 6B), and it was lower in highacid fruits than in low-acid fruits. Further correlation analysis indicated that the fruit weight loss was significantly negatively correlated with the TA content during storage in No. 168, 080,

fruits throughout the storage period (Figure 5B). Correspondingly, compared with low-acid fruits, the expression of NADisocitrate dehydrogenase (NAD-IDH) was significantly lower in high-acid fruits at 60 and 85 DAS (Figure 5C); and the expression of 2-oxoglutarate dehydrogenase (2-OGDH) was significantly lower in high-acid fruits at 20 and 40 DAS (Figure 5D). Moreover, GABA shunt genes, including GAD1 and GABP, showed an obviously lower expression in high-acid fruits than in low-acid fruits at 40, 60, and 85 DAS (Figure 5E and F); the expression of GABA-T was significantly lower in high-acid fruits mainly at 20, 60, and 85 DAS (Figure 5G); for SSADH, its expression was only significantly lower in high-acid fruits at 60 DAS, and no obvious differences were observed between No.168 and high-acid fruits at other storage stages (Figure 5H). In general, the expression activity of GABA shunt together with TCA cycle related genes including m-ACO, NAD-IDH, and 2OGDH was obviously lower in high-acid fruits than low-acid fruits during postharvest storage. In addition, the expression of H+-ATPase Isoform 10 (AHA10) and AA-TFP was also significantly lower in highacid fruits compared with in low-acid fruits during storage, 1673


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Journal of Agricultural and Food Chemistry and 181 (R = −0.9132, p < 0.05; R = −0.8756, p < 0.05; R = −0.9021, p < 0.05, respectively) (Table 1).

apple,31 and peach,32 but not for organic acids. In fact, a previous study showed a high heritability value of 0.53 for organic acids in strawberry, indicating that organic acid level is mainly under genetic control, but no published data are available for comparison.33 In our study, the TA content slightly fluctuated in both the parents and hybrid population from 2012 to 2014, but such fluctuation did not change the transgressive character of the hybrid (Figure 1A and Figure S1). In addition, the storage experiment of the selected fruits showed that the high acid feature of the high-acid fruits was maintained through the whole storage period (Figure 3A). These results suggest that although organic acid content is influenced by environment to some extent, it is mainly controlled by heredity. Effect of GABA Pathway Activity on Citrate Level. As the major organic acid in citrus fruit, citrate shows varying contents in different citrus varieties. Citrate is accumulated during fruit development and is decreased during fruit ripening and postharvest storage in citrus fruit.6,8 GABA shunt is one of major pathways participating in citrate utilization during fruit ripening.7,8 Our results showed that the genes in GABA pathway generally exhibited obvious low expression in high-acid fruits compared with in low-acid fruits during postharvest storage (Figure 5E−H), which can largely explain the high-acid trait of the high-acid fruits during postharvest storage (Figure 3A). Previous studies have suggested that the inhibition of mACO creates a metabolic block in the TCA cycle, which leads to citrate accumulation in sour lemon fruit.11 Here, we found that the expression of m-ACO was significantly lower in highacid fruits during storage (Figure 5B). Other genes in TCA cycle, including NAD-IDH and 2-OGDH, also showed low expression in high-acid fruits (Figure 5C and D). The block in TCA cycle may also partially explain the high citrate accumulation in high-acid fruits. In addition, we found that TT8, an important regulator of flavonoid biosynthesis pathway,34 had obviously high expression in high-acid fruits (Figure

Table 1. Pearson’s Correlation Analysis between Weight Loss, TA, and TSS

a

No. 130

weight loss

TA

TSS

weight loss TA TSS No. 168

1 0.2879 0.6383 weight loss

1 0.0872 TA

1 TSS


1 −0.9132b 0.0000 weight loss

1 0.3305 TA

1 TSS


1 −0.8756b 0.2373 weight loss

1 0.2366 TA

1 TSS

weight loss TA TSS

1 −0.9021b 0.7197

1 0.3807

1

P < 0.01. bP < 0.05.

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DISCUSSION Genetic Effects on Organic Acid Level. In our study, a hybrid population was obtained by the cross of HB with FC conducted ten years ago in order to analyze the genetic effect on organic acid level in citrus fruit. Although it seems reasonable to expect that the hybrid would show a TA level intermediate between its parents, the results revealed that the TA content in the hybrid population was transgressive (Figure 1A and Figure S1), which could be attributed to the accumulation of citrate (data not shown). Transgressive behavior has been observed for volatile compounds in citrus,30

Figure 7. Sketch of the major pathways involved in high-acid trait. The green circle indicates that the metabolite content or gene expression level is significantly lower in high-acid fruits (No. 080 and 181) than low-acid fruits (No. 130 and 168). The red circle indicates that the metabolite content or gene expression level is significantly higher in high-acid fruits than low-acid fruits. 1674


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5K). This result is in conformity with the results of a previous study, which suggested that TT8 is positively correlated with acid level, and it is possible that flavonoid metabolism may play a role in controlling acid accumulation in citrus fruit.18 According to the results of previous studies7,10 and the present study, we established a model to explain the acid accumulation in high-acid fruits (Figure 7). As other genes related to citrate metabolism showed no specific expression patterns in high- and low-acid fruits, we did not present the corresponding results in Figure 7. Organic Acids and Fruit Water Loss. Previous studies have shown that granulation (also called dryness with little extractable juice) causes a decrease in TA (especially citrate) and an increase of pH in the juice sacs of various citrus species and cultivars.35,36 Furthermore, there have been increasing evidence suggesting that cytosolic pH could regulate water transport through the gating of aquaporins.37 As we know, the supply of water to fruit from the tree is cut off after fruit harvest, and the original water sink turns into water source, which leads to the release of water from the fruit to the environment. Cytosolic acidification could result in protein protonation of plant aquaporins and closure of the aquaporins in the plasma membrane.38 Our results indicated that the fruit weight loss (mainly represented by water loss)39 was lower in high-acid fruits than in low-acid fruits during storage (Figure 6B). Correlation analysis showed that TA level was significantly negatively correlated with fruit weight loss in No. 168, 080, and 181 during storage (Table 1). These results imply a close relationship between organic acids and water loss in postharvest citrus fruit. Further research should be carried out to obtain more insights into the specific relationship between organic acid and water metabolism in citrus fruit. In conclusion, this study reveals that the importance of genetic effect on organic acid level in postharvest citrus fruit. Our results indicate that the high acid accumulation in postharvest high-acid fruits can be attributed to the low GABA pathway activity and block of TCA cycle by low expression of m-ACO. We also found that during postharvest storage, there is a negative correlation between TA level and fruit weight loss, two parameters closely connected to the fruit quality and storage performance. Taken together, our results could contribute to the development of effective ways to control acid content in citrus production.

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This work was supported by Huazhong Agricultural University Scientific & Technological Self-innovation Foundation, the Natural Science Foundation of China (NSFC; No. 31221062, 31572176); the National Science & Technological Pillar Program of China (No. 2015BAD16B06); Citrus Postharvest Biology Foundation and Storage Technology (No. 2014PY014); the National Foundation Innovation Team, the Project of Excellent Scientist Fund in Hubei (No. 31521092); Huazhong Agricultural University Special Fund for Major Project of Independent Innovation and Science & Technological Pillar Program of Huber Provence. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Zuoxiong Liu (Foreign Language College of Huazhong Agricultural University) for modifying this manuscript.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b05237. Fruit weight (g), fruit shape index, pericarp thickness (mm), color index (1000a/L/b), TSS (%), and VC (mg/ 100g FW) in individuals of HB (Citrus grandis) × Fairchild (Citrus reticulata) hybrid population; specific primers used in real-time reverse transcriptase-PCR; TA contents in HB × Fairchild hybrid population in 2012 (A) and 2013 (B) (PDF)

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Corresponding Author

*E-mail: [email protected]; Tel.: +86 2787281796; Fax: +86 2787280622. ORCID

Ling Sheng: 0000-0001-7593-9627 1675


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GABA Pathway Rate-Limit Citrate Degradation in Postharvest Citrus

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